Template-fixed peptidomimetics

ABSTRACT

The template-fixed β-hairpin peptidomimetics Cyclo(-Tyr-His-Cys-Ser-Ala- D Pro-Dab-Arg-Tyr-Cys-Tyr-Gln-Lys- D -Pro-Pro), disulfide bond between Cys4 and Gysl1, and pharmaceutically acceptable salts thereof, with X being Ala or Tyr, have CXCR4 antagonizing properties and can be used for where cancer is mediated or resulting from CXCR4 receptor activity

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a Continuation of U.S. application Ser. No. 15/379,820, filed onDec. 15, 2016, which is a continuation of U.S. application Ser. No.14/555,796, filed Nov. 28, 2014, now U.S. Pat. No. 9,556,234, which is acontinuation of U.S. application Ser. No. 13/764,099, filed Feb. 11,2013, now U.S. Pat. No. 8,921,325, which is a continuation of U.S.application Ser. No. 12/528,926, filed Oct. 27, 2009, now U.S. Pat. No.8,399,611, which is the National Stage of International application no.PCT/CH2007/00101, filed Feb. 28, 2007, of which all of the disclosuresare incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The present invention provides template-fixed β-hairpin peptidomimeticswhich are having CXCR4 antagonizing activity and are embraced by thegeneral disclosure of, but not specifically disclosed in WO2004/096840A1.

SUMMARY OF THE INVENTION

The 13-hairpin peptidomimetics of the invention areCyclo(-Tyr-His-X-Cys-Ser-Ala-^(D)Pro-Dab-Arg-Tyr-Cys-Tyr-Gln-Lys-^(D)Pro-Pro),disulfide bond between Cys4 and Cysl 1, and pharmaceutically acceptablesalts thereof, with X being Ala or Tyr.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Increase in Colony Forming Units per ml of blood (CFU-GM) overtime for both the compound of Example I (5 mg) and the referencecompound AMD3100.

FIG. 2. Increase in Colony Forming Units per ml of blood (CFU-GM) overtime for both the compound of Example 2 and the reference compoundAMD3100.

FIG. 3. Average number of CD34(+) cells per μL, of blood over time forcompound of Example 1.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the invention, the aforesaid β-hairpin mimetics andpharmaceutically acceptable salts thereof can be prepared by a processwhich comprises

(a) coupling an appropriately functionalized solid support with anappropriately N-protected derivative of Pro;(b) removing the N-protecting group from the product obtained in step(a);(c) coupling the product thus obtained with an appropriately N-protectedderivative of ^(D)Pro;(d) removing the N-protecting group from the product thus obtained;(e) coupling the product thus obtained with an appropriately N-protectedderivative of the amino acid which in the desired end-product is inposition 14, i.e. Lys, the amino group present in its side-chain beinglikewise appropriately protected;(f) removing the N-protecting group from the product thus obtained;(g) effecting steps substantially corresponding to steps (e) and (f)using appropriately N-protected derivatives of the amino acids which inthe desired end-product are in positions 13 to 1, i.e. Gln, Tyr, Cys,Tyr, Arg, Dab, ^(D)Pro, Ala, Ser, Cys, Ala or Tyr, His and Tyr, anyfunctional group which may be present in said N-protected amino acidderivatives being likewise appropriately protected;(h) forming the disulfide β-strand linkage between the side-chains ofthe Cys residues in positions 4 and 11;(i) detaching the product thus obtained from the solid support;(j) cyclizing the product cleaved from the solid support;(k) removing any protecting groups present on functional groups of anymembers of the chain of amino acid residues; and(l) if desired, converting the product thus obtained into apharmaceutically acceptable salt or converting a pharmaceuticallyacceptable, or unacceptable, salt thus obtained into the correspondingfree compound or into a different, pharmaceutically acceptable, salt.

The steps of the aforesaid process can be carried out by methods whichare well known to any person adequately skilled in peptide chemistry.

The β-hairpin peptidomimetics of the invention can be used in a widerange of applications for preventing HIV infections in healthyindividuals or for slowing and halting viral progression in infectedpatients; or where cancer is mediated or resulting fromCXCR4 receptoractivity; or where immunological diseases are mediated or resulting fromCXCR4 receptor activity; or for treating immuno suppression; or fortreating inflammation, or, in particular, for stem cell mobilisation ofperipheral blood stem cells and/or mesenchymal stem cell (MSC) and/orother stem cells which retention depend on the CXCR4-receptor.

The β-hairpin peptidomimetics of the invention may be administered perse or may be applied as an appropriate formulation together withcarriers, diluents or excipients well known in the art.

In particular, the β-hairpin peptidomimetics of the invention can beused as a treatment to increase hematopoetic stem cell (HSC) releasefrom the bone marrow to be used in allogenic or autologous transplant.

The acute treatment with infused HSC is widely used to restore immunefunctions in patients who have received myeloablative therapy during thetreatment of malignancies such as multiple myeloma and non-Hodgkin'slymphoma. Patients or donors are treated with the HCS mobilisationagent, such as a compound of the invention, and the cells aresubsequently collected from peripheral blood by apharesis. HCS aretransplanted back after e.g. chemotherapy treatment into the patient(autologous transplant) or from donor to recipient (allogenictransplant), thus promoting the restoration of immune function (Frúhaufet al., Br. J. Haematol. 122, 360-375 (2003)).

Other applications of the HSC treatment include, but are not limited to,therapeutic angiogenesis in case of e.g. heart attack (Shepherd R M etal, Blood 2006 108(12):3662-3667).

The β-hairpin peptidomimetics of the invention may also be used to treator prevent HIV infections or cancer such as breast cancer, brain cancer,prostate cancer, lung cancer, kidney cancer, neuroblastoma,non-Hodgkin's lymphoma, ovarian cancer, multiple myeloma, chroniclyphomphocytic leukemia, pancreatic cancer, melanoma, angiogenesis andhaematopoetic tissues; or inflammatory disorders such as asthma,allergic rhinitis, hypersensitivity lung diseases, hypersensitivitypneumonitis, eosinophilic pneumonias, delayed-type hypersensitivity,interstitial lung disease (ILD), idiopathic pulmonary fibrosis, ILDassociated with rheumatoid arthritis, systemic lupus erythematosus,ankylosing spondylitis, peripheral vascular disease, systemic sclerosis,Sjogren's syndrome, von Hippel Lindau disease, systemic anaphylaxis orhypersensitivity responses, drug allergies, rheumatoid arthritis,psoriatic arthritis, Behcet's Syndrome, mucositis, Crohn's disease,multiple sclerosis, myasthenia gravis, juvenile onset diabetes,glomerulonephritis, autoimmune throiditis, graft rejection, includingallograft rejection or graft-versus-host disease, inflammatory boweldiseases, inflammatory dermatoses; or to treat immunosuppression,including immunosuppression induced by graft/transplantation rejection.

The β-hairpin peptidomimetics of the invention can be administeredsingly, as mixtures of more than one β-hairpin peptidomimetics, incombination with, as the case may be, other HSC mobilisation agents, oranti-HIV agents, or antimicrobial agents, or anti cancer agents, oranti-inflammatory agents, and/or in combination with otherpharmaceutically active agents.

Pharmaceutical compositions comprising β-hairpin peptidomimetics of theinvention may be manufactured by means of conventional mixing,dissolving, granulating, coated tablet-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes. Pharmaceuticalcompositions may be formulated in conventional manner using one or morephysiologically acceptable carriers, diluents, excipients orauxilliaries which facilitate processing of the active β-hairpinpeptidomimetics into preparations which can be used pharmaceutically.Proper formulation depends upon the method of administration chosen.

For topical administration the β-hairpin peptidomimetics of theinvention may be formulated as solutions, gels, ointments, creams,suspensions, etc. as are well-known in the art.

Systemic formulations include those designed for administration byinjection, e.g. subcutaneous, intravenous, intramuscular, intrathecal orintraperitoneal injection, as well as those designed for transdermal,transmucosal, oral or pulmonary administration.

For injections, the β-hairpin peptidomimetics of the invention may beformulated in adequate solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological saline buffer. The solutions may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Alternatively, the β-hairpin peptidomimetics of the invention may be inpowder form for combination with a suitable vehicle, e.g., sterilepyrogen-free water, before use.

For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation as known in the art.

For oral administration, the compounds can be readily formulated bycombining the active β-hairpin peptidomimetics of the invention withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the β-hairpin peptidomimetics of the invention to beformulated as tablets, pills, dragees, capsules, liquids, gels, syrups,slurries, suspensions etc., for oral ingestion by a patient to betreated. For oral formulations such as, for example, powders, capsulesand tablets, suitable excipients include fillers such as sugars, e. g.lactose, sucrose, mannitol and sorbitol; cellulose preparations such asmaize starch, wheat starch, rice starch, potato starch, gelatin, gumtragacanth, methyl cellulose, hydroxypropylmethyl cellulose, sodiumcarboxymethylcellulose; granulating agents; and binding agents. Ifdesired, desintegrating agents may be added, such as cross-linkedpolyvinylpyrrolidones, agar, or alginic acid or a salt thereof, such assodium alginate. If desired, solid dosage forms may be sugar-coated orenteric-coated using standard techniques.

For oral liquid preparations such as, for example, suspensions, elixirsand solutions, suitable carriers, excipients or diluents include water,glycols, oils, alcohols, etc. In addition, flavoring agents,preservatives, coloring agents and the like may be added.

For buccal administration, the composition may take the form of tablets,lozenges, etc. formulated as usual.

For administration by inhalation, the β-hairpin peptidomimetics of theinvention are conveniently delivered in form of an aeorosol spray frompressurized packs or a nebulizer, with the use of a suitable propellant,e.g. dichlorodifluoromethane, trichlorofluromethane, carbon dioxide oranother suitable gas. In the case of a pressurized aerosol the dose unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the β-hairpinpeptidomimetics of the invention and a suitable powder base such aslactose or starch.

The compounds may also be formulated in rectal or vaginal compositionssuch as suppositories together with appropriate suppository bases suchas cocoa butter or other glycerides.

In addition to the formulations described previously, the β-hairpinpeptidomimetics of the invention may also be formulated as depotpreparations. Such long acting formulations may be administered byimplantation (e.g. subcutaneously or intramuscularly) or byintramuscular injection. For the manufacture of such depot preparationsthe β-hairpin peptidomimetics of the invention may be formulated withsuitable polymeric or hydrophobic materials (e.g. as an emulsion in anacceptable oil) or ion exchange resins, or as sparingly soluble salts.

In addition, other pharmaceutical delivery systems may be employed suchas liposomes and emulsions well known in the art. Certain organicsolvents such as dimethylsulfoxide may also be employed. Additionally,the β-hairpin peptidomimetics of the invention may be delivered using asustained-release system, such as semipermeable matrices of solidpolymers containing the therapeutic agent. Various sustained-releasematerials have been established and are well known by those skilled inthe art. Sustained-release capsules may, depending on their chemicalnature, release the compounds for a few weeks up to over 100 days.Depending on the chemical nature and the biological stability of thetherapeutic agent, additional strategies for protein stabilization maybe employed.

As the β-hairpin pepdidomimetics of the invention contain chargedresidues, they may be included in any of the above-describedformulations as such or as pharmaceutically acceptable salts.Pharmaceutically acceptable salts tend to be more soluble in aqueous andother protic solvents than are the corresponding free forms.Particularly suitable pharmaceutically acceptable salts include saltswith, carboxylic, phosphonic, sulfonic and sulfamic acids, for exampleacetic acid, propionic acid, octanoic acid, decanoic acid, dodecanoicacid, glycolic acid, lactic acid, fumaric acid, succinic acid, adipicacid, pimelic acid, suberic acid, azelaic acid, malic acid, tartaricacid, citric acid, amino acids, such as glutamic acid or aspartic acid,maleic acid, hydroxymaleic acid, methylmaleic acid,cyclohexanecarboxylic acid, adamantanecarboxylic acid, benzoic acid,salicylic acid, 4-aminosalicylic acid, phthalic acid, phenylacetic acid,mandelic acid, cinnamic acid, methane- or ethane-sulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 2-naphthalenesulfonic acid,1,5-naphthalene-disulfonic acid, 2-, 3- or 4-methylbenzenesulfonic acid,methylsulfuric acid, ethylsulfuric acid, dodecylsulfuric acid,N-cyclohexylsulfamic acid, N-methyl-, N-ethyl- or N-propyl-sulfamicacid, and other organic protonic acids, such as ascorbic acid. Suitableinorganic acids are, for example, hydrohalic acids, such as hydrochloricacid, sulfuric acid, and phosphoric acid.

The n-hairpin peptidomimetics of the invention, in free form or in theform of pharmaceutically acceptable salts, or compositions thereof, willgenerally be used in an amount effective to achieve the intendedpurpose. It is to be understood that the amount used will depend on aparticular application.

For topical administration to treat or prevent HIV infections atherapeutically effective dose can be determined using, for example, thein vitro assays provided in the examples. The treatment may be appliedwhile the HIV infection is visible, or even when it is not visible. Anordinary skilled expert will be able to determine therapeuticallyeffective amounts to treat topical HIV infections without undueexperimentation.

For systemic administration, a therapeutically effective dose can beestimated initially from in vitro assays. For example, a dose can beformulated in animal models to achieve a circulating β-hairpinpeptidomimetic concentration range that includes the IC₅₀ as determinedin the cell culture (i.e. the concentration of a test compound that islethal to 50% of a cell culture). Such information can be used to moreaccurately determine useful doses in humans.

Initial dosages can also be determined from in vivo data, e.g. animalmodels, using techniques that are well known in the art. One havingordinary skill in the art could readily optimize administration tohumans based on animal data.

Dosage amounts for applications as anti-HIV agents may be adjustedindividually to provide plasma levels of the ft-hairpin peptidomimeticsof the invention which are sufficient to maintain the therapeuticeffect. Therapeutically effective serum levels may be achieved byadministering multiple doses each day.

In cases of local administration or selective uptake, the effectivelocal concentration of the n-hairpin peptidomimetics of the inventionmay not be related to plasma concentration. One having the ordinaryskill in the art will be able to optimize therapeutically effectivelocal dosages without undue experimentation.

The amount of β-hairpin peptidomimetics administered will, of course, bedependent on the subject being treated, on the subject's weight, theseverity of the affliction, the manner of administration and thejudgement of the prescribing physician.

The anti-HIV therapy may be repeated intermittently while infections aredetectable or even when they are not detectable. The therapy may beprovided alone or in combination with other drugs, such as for exampleother anti-HIV agents or anti cancer agents, or other antimicrobialagents.

Normally, a therapeutically effective dose of the β-hairpinpeptidomimetics described herein will provide therapeutic benefitwithout causing substantial toxicity.

Toxicity of then β-hairpin peptidomimetics of the invention can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., by determining the LD₅₀ (the dose lethal to50% of the population) or the LD₁₀₀ (the dose lethal to 100% of thepopulation). The dose ratio between toxic and therapeutic effect is thetherapeutic index. Compounds which exhibit high therapeutic indices arepreferred. The data obtained from these cell culture assays and animalstudies can be used in formulating a dosage range that is not toxic foruse in humans. The dosage of the β-hairpin peptidomimetics of theinvention lies preferably within a range of circulating concentrationsthat include the effective dose with little or no toxicity. The dosagemay vary within the range depending upon the dosage form employed andthe route of administration utilized. The exact formulation, route ofadministration and dose can be chosen by the individual physician inview of the patient's condition (see, e.g. Fingl et al. 1975, in: ThePharmacological Basis of Therapeutics, Ch. 1, p. 1).

The following Examples illustrate the invention in more detail but arenot intended to limit its scope in any way. The following abbreviationsare used in these Examples:

-   -   HBTU: 1-benzotriazol-1-yl-tetramethyluronium hexafluorophosphate        (Knorr et al. Tetrahedron Lett. 1989, 30, 1927-1930);    -   HOBt: 1-hydroxybenzotriazole;    -   DMA: diisopropylethylamine;    -   HOAT: 7-aza-1-hydroxybenzotriazole;    -   HATU: O-(7-aza-benzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium        hexafluorophosphate (Carpino et al. Tetrahedron Lett. 1994, 35,        2279-2281).

1. Peptide Synthesis Coupling of the First Protected Amino Acid Residueto the Resin

0.5 g of 2-chlorotritylchloride resin (100-200 mesh, copoly(styrene-1%DVB) polymer matrix, Cat. No. 01-64-0114, Novabiochem, Merck BiosciencesLtd.) (Barlos et al. Tetrahedron Lett.

1989, 30, 3943-3946) (1.4 mMol/g, 0.7 mmol) was filled into a driedflask. The resin was suspended in CH₂Cl₂ (2.5 ml) and, allowed to swellat room temperature under constant stirring for 30 min. The resin wastreated with 0.49 mMol (0.7 eq) of the first suitably protected aminoacid residue and 488 μl (4 eq) of diisopropylethylamine (DIEA) in CH₂Cl₂(2.5 ml), the mixture was shaken at 25° C. for 4 hours. The resin wasshaken (CH₂Cl₂/MeOH/DIEA: 17/2/1), 30 ml for 30 min; then washed in thefollowing order with CH₂Cl₂ (1×), DMF (1×), CH₂Cl₂ (1×), MeOH (1×),CH₂Cl₂ (1×), MeOH (1×), CH₂Cl₂ (2×), Et₂O (2×) and dried under vacuumfor 6 hours.

Loading was typically 0.6-0.9 mMol/g.

The following preloaded resin was prepared:Fmoc-Pro-2-chlorotritylresin.

Synthesis of the Fully Protected Peptide Fragment

The synthesis was carried out on a Syro-peptide synthesizer(MultiSynTech GmbH) using 24 to 96 reaction vessels. In each vessel wereplaced approximately 60 mg (weight of the resin before loading) of theabove resin. The following reaction cycles were programmed and carriedout:

Step Reagent Time 1 CH₂Cl₂, wash and swell (manual) 1 × 3 min. 2 DMF,wash and swell 1 × 60 min.  3 40% piperidine/DMF 2 × 5 min. 4 DMF, wash5 × 1 min. 5 5 equiv. Fmoc amino acid/DMF + 2 × 60 min.  5 eq. HBTU + 10eq. DIEA 6 DMF, wash 5 × 1 min. 7 40% piperidine/DMF 2 × 5 min. 8 DMF,wash 5 × 1 min. 9 CH₂Cl₂, wash (at the end of the synthesis) 3 × 1 min.

Steps 3 to 6 are repeated to add each amino-acid.

Analytical Method:

Analytical HPLC retention times (RT, in minutes) were determined using aJupiter Proteo 90 A column, 150×2.0 mm, (cod. 00F-4396-B0-Phenomenex)with the following solvents A (H₂O+0.1% TFA) and B (CH₃CN+0.1% TFA) andthe gradient: 0 min: 95% A, 5% B; 0.5 min: 95% A, 5% B; 20 min: 40% A,60% B; 21 min: 0% A, 100% B; 23 min: 0% A, 100% B; 23.1 min: 95% A, 5%B; 31 min: 95% A, 5% B.

Formation of Disulfide β-Strand Linkage

After completion of the synthesis, the resin was swelled in 3 ml of dryDMF for 1 h. Then 10 eq. of iodine solution in DMF (6 ml) were added tothe reactor, followed by stirring for 1.5 h. The resin was filtered anda fresh solution of iodine (10 eq.) in DMF (6 nil) was added, followedby stirring for another 3 h. The resin was filtered and washed with DMF(3×) and CH₂Cl₂ (3×).

Cleavage, Backbone Cyclization, Deprotection and Purification of thePeptide

After formation of the disulfide β-strand linkage, the resin wassuspended in 1 ml (0.14 mMol) of 1% TFA in CH₂Cl₂ (v/v) for 3 minutesand filtered, and the filtrate was neutralized with 1 ml (1.15 mMol) of20% DIEA in CH₂Cl₂ (v/v). This procedure was repeated twice to ensurecompletion of the cleavage. The resin was washed three times with 1 mlof CH₂Cl₂. The CH₂Cl₂ layer was evaporated to dryness.

The volatiles were removed and 8 ml dry DMF were added to the tube. Then2 eq. of HATU in dry DMF (1 ml) and 4 eq. of DIPEA in dry DMF (1 ml)were added to the peptide, followed by stirring for 16 h. The volatileswere evaporated to dryness. The crude cyclised peptide was dissolved in7 ml of CH₂Cl₂ and extracted with 10% acetonitrile in H₂O (4.5 ml) threetimes. The CH₂Cl₂ layer was evaporated to dryness. To deprotect thepeptide fully, 3 ml of cleavage cocktail TFA:TIS:H₂O (95:2.5:2.5) wereadded, and the mixture was kept for 2.5 h. The volatiles were evaporatedto dryness and the crude peptide was dissolved in 20% AcOH in water (7ml) and extracted with isopropyl ether (4 ml) for three times. Theaqueous layer was collected and evaporated to dryness, and the residuewas purified by preparative reverse phase HPLC.

After lyophilisation the products were obtained as white powders andanalysed by the HPLC-ESI-MS analytical method described above. Theanalytical data comprising purity after preparative HPLC and ESI-MS aregiven.

Example 1

The peptide was synthesized starting with the amino acid L-Pro which wasgrafted to the resin. Starting resin was Fmoc-Pro-2-chlorotrityl resin,which was prepared as described above. The linear peptide wassynthesized on solid support according to the procedure described abovein the following sequence:Resin-Pro-^(D)Pro-Lys-Gln-Tyr-Cys-Tyr-Arg-Dab-^(D)Pro-Ala-Ser-Cys-Ala-His-Tyr.A disulfide β-strand linkage was introduced as described above. Theproduct was cleaved from the resin, cyclized, deprotected and purifiedas indicated by preparative reverse phase LC-MS.

After lyophilisation the product was obtained as white powder andanalysed by the HPLC-ESI-MS analytical method described above ([M+2H]²⁺:933.1; RT: 10.47; UV-purity: 72%).

Example 2

The peptide was synthesized starting with the amino acid L-Pro which wasgrafted to the resin. Starting resin was Fmoc-Pro-2-chlorotrityl resin,which was prepared as described above. The linear peptide wassynthesized on solid support according to the procedure described abovein the following sequence:Resin-Pro-^(D)Pro-Lys-Gln-Tyr-Cys-Tyr-Arg-Dab-^(D)Pro-Ala-Ser-Cys-Tyr-His-Tyr.A disulfide β-strand linkage was introduced as described above. Theproduct was cleaved from the resin, cyclized, deprotected and purifiedas indicated by preparative reverse phase LC-MS. After lyophilisationthe product was obtained as white powder and analysed by the HPLC-ESI-MSanalytical method described above ([M+2H]²⁺: 978.6; RT: 10.95;UV-purity: 82%).

2. Biological Methods 2.1. Preparation of the Peptides.

Lyophilized peptides were weighed on a Microbalance (Mettler MT5) anddissolved in sterile water to a final concentration of 1 mM unlessstated otherwise. Stock solutions were kept at +4° C., light protected.

2.2. Ca²⁺⁻ Assay: CXCR4-Antagonizing Activity of the Peptides.

Increases in intracellular calcium were monitored using a Flexstation384 (Molecular Devices, Sunnyvale, Calif.) to assay the peptides forCXCR4 antagonism in a mouse pre-B cell line 300-19 stably transfectedwith human CXCR4 [see references 1, 2 and 3, below]. The cells werebatch loaded with the Calcium 3 Assay kit (Molecular Devices) in assaybuffer (Hanks Balanced salt solution, HBSS, 20 mM HEPES, pH 7.4, 0.1%BSA) for 1 h at room temperature and then 200,000 labeled cells weredispensed into black 96 well assays plates (Costar No. 3603). A 20-foldconcentrated solution of peptide in assay buffer was added to the cellsand the whole plate was centrifuged to settle the cells to the bottom ofthe wells. Calcium mobilization induced by 10 nM stromal-derivedfactor-1 (SDF-1) was measured in the Flexstation 384 (excitation, 485nM; emission, 525 nM) for 90 seconds. A maximal change in fluorescenceresponse above baseline was used to calculate antagonist activity. Thedata for dose response curves (antagonist concentration versus % maximumresponse) were fitted to a four parameter logistic equation usingSoftmaxPro 4.6 (Molecular Devices), from which IC₅₀% values werecalculated.

2.3. FIGS-Assay™

The assay was performed according to ref. 5, below. Stock dilutions ofthe peptides (10 μM) were prepared by dissolving in 10 μM Tris-HCl atroom temperature. Stock solutions were kept at +4° C., light protected.Working dilutions were prepared extemporaneously by serial dilution inPhosphate Buffered Saline (PBS) and added in a final volume of 10 μldirectly to the cell cultures. After 48 hours of co-cultivation thecultures were rinsed with PBS and then exposed toglutaraldehyde/formaldehyde (0.2%/2%) in PBS for five minutes. Forphotometric quantification the fixed cultures were subsequentlyincubated with ortho-nitro-phenyl-galactopyranoside (ONPG) as aβ-galactosidase substrate, which was enzymatically converted into thechromophore ortho-nitrophenol (ONP). The read out is directly obtainedby measuring optical density of wells at 405 nm in an iEMS 96 well-platereader.

2.4. Cytotoxicity Assay

The cytotoxicity of the peptides to HFLA cells (Acc57) and COS-7 cells(CRL-1651) was determined using the MTT reduction assay [see ref. 6 and7, below]. Briefly the method was as follows: BELA cells and COS-7 cellswere seeded at 7.0×10³ and, respectively, 4.5×10³ cells per well andgrown in 96-well microtiter plates for 24 hours at 37° C. at 5% CO₂. Atthis point, time zero (Tz) was determined by MTT reduction (see below).The supernatant of the remaining wells was discarded, and fresh mediumand the peptides in serial dilutions of 12.5, 25 and 50 μM were pipettedinto the wells. Each peptide concentration was assayed in triplicate.Incubation of the cells was continued for 48 hours at 37° C. at 5% CO₂.Wells were then washed once with PBS and subsequently 100 μl MTT reagent(0.5 mg/mL in medium RPMI1640 and, respectively, DMEM) was added to thewells. This was incubated at 37° C. for 2 hours and subsequently themedium was aspirated and 100 μl isopropanol was added to each well. Theabsorbance at 595 nm of the solubilized product was measured(OD₅₉₅peptide). For each concentration averages were calculated fromtriplicates. The percentage of growth was calculated as follows:(OD₅₉₅peptide-OD₅₉₅Tz-OD₅₉₅Empty well)/(OD₅₉₅Tz-OD₅₉₅Empty well)×100%and was plotted for each peptide concentration.

The LC 50 values (Lethal Concentration, defined as the concentrationthat kills 50% of the cells) were determined for each peptide by usingthe trend line function of EXCEL (Microsoft Office 2000) for theconcentrations (50, 25, 12.5 and 0 μM), the corresponding growthpercentages and the value −50, (=TREND(C50:CO₃%50:%0,−50)). The GI 50(Growth Inhibition) concentrations were calculated for each peptide byusing a trend line function for the concentrations (50, 25, 12.5 and 0μg/ml), the corresponding percentages and the value 50, (=TREND(C₅₀:C₀,%₅₀:%₀,50).

2.5. Cell Culture

‘CCR5’ cells were cultured in DMEM medium with 4500 mg/ml glucose, 10%fetal bovine serum (FBS), supplemented with 50 U/ml Penicillin and 50μg/ml Streptomycin (Pen/Strept.). Hut/4-3 cells were maintained in RPMImedium, 10% FBS, supplemented with Pen/Strept. and 10 mM HEPES. HFLAcells and CCRF-CEM cells were maintained in RPMI1640 plus 5% FBS,Pen/Strept and 2 mM L-Glutamine. Cos-7 cells were grown in DMEM mediumwith 4500 mg/ml glucose supplemented with 10% FCS, Pen/Strept. and 2 mML-Glutamine. All cell lines were grown at 37° C. at 5% CO₂. Cell media,media supplements, PBS-buffer, HEPES, Pen/Strept., L-Glutamine and serawere purchased from Gibco (Pailsey, UK). All fine chemicals came fromMerck (Darmstadt, Germany).

2.6. Hemolysis

The peptides were tested for their hemolytic activity against human redblood cells (hRBC). Fresh hRBC were washed three times with phosphatebuffered saline (PBS) by centrifugation for 10 min at 2000×g. Peptidesat a concentration of 100 μM were incubated with 20% v/v hRBC for 1 hourat 37° C. The final erythrocyte concentration was approximately 0.9×10⁹cells per ml. A value of 0% resp. 100% cell lysis was determined byincubation of the hRBC in the presence of PBS alone and respectively0.1% Triton X-100 in H₂O. The samples were centrifuged and thesupernatant was 20-fold diluted in PBS buffer and the optical density(OD) of the sample at 540 nM was measured. The 100% lyses value(OD₅₄₀H₂O) gave an OD₅₄₀ of approximately 1.3-1.8. Percent hemolysis wascalculated as follows: (OD₅₄₀peptide/OD₅₄₀H₂0)×100%.

2.7. Chemotactic Assay (Cell Migration Assay)

The chemotactic response of CCRF-CEM cells to a gradient of stromalcell-derived factor 1α (SDF-1) was measured using disposable assayplates from Neuroprobe (5μ pore size) (Gaithersburg, Md.), according tothe manufacturer's directions and references therein [especially ref. 8,below]. Briefly, one 175 cm³ flask was washed once with Dubecco'sphosphate buffered saline (DPBS), and trypsinized for 10 minutes oruntil cells had lifted. The trypsin was neutralized by the addition offresh medium containing serum and the cells were pelleted, washed oncein DPBS, and resuspended at 1-0.5×10⁷ cells/rill in RPMI+0.5% bovineserum albumin (BSA). 45 μl of cell suspension were mixed with 5 μl of10-fold concentrated PEM peptide diluted in the same assay medium. 35 μlof this mixture were applied to the top of the assay filter. The cellswere allowed to migrate (at 37′) into the bottom chamber of the assayplate containing 1 nM SDF-1. After 4 hours, the filter was removed andMTT was added to the migrated cells to a final concentration of 0.5mg/ml, and incubated for a further 4 hours. After labeling with MTT, allmedium was removed and 100 μl of isopropanol+10 mM HCl were added to thecells. The optical absorbance at 595 nm (ABS₅₉₅) was read using a TecanGenios plate reader with Magellan software. The number of cells migratedwas determined by comparing ABS₅₉₅ values against a standard curvegenerated with a known number of cells in the assay plate and wereplotted against SDF-1 concentration to obtain a sigmoidal curve and todetermine the IC₅₀ values. The values for IC50 were determined using theTrendline function in Microsoft Excel by fitting a logarithmic curve tothe averaged datapoints.

2.8 Plasmastability

405 μl of plasma/albumin solution were placed in a polypropylene (PP)tube and spiked with 45 μl of compound from a 100 μNI solution B,derived from 135 μl of PBS and 15 μl of 1 mM peptide in PBS, pH 7.4. 150μl aliquots were transferred into individual wells of the 10 kDa filterplate (Millipore MAPPB 1010 Biomax membrane). For “0 minutes controls”:270 μd of PBS were placed in a PP tube and 30 μl of stock solution B wasadded and vortexed. 150 μl of control solution was placed into one wellof the filter plate and serves as “filtered control”.

Further 150 μl of control solution were placed directly into a receiverwell (reserved for filtrate) and serve as “not-filtered control”. Theentire plate including evaporation lid was incubated for 60 min at 37°C. Plasma samples (rat plasma: Harlan Sera lab UK, human plasma:Blutspendezentrum Zürich) were centrifuged at least for 2 h at 4300 rpm(3500 g) and 15° C. in order to yield 100 μl filtrate. For “serumalbumin”-samples (freshly prepared human albumin: Sigma A-4327, ratalbumin: Sigma A-6272, all at 40 mg/ml concentration in PBS)approximately 1 hour of centrifugation is sufficient. The filtrates inthe receiver PP plate were analysed by LC/MS as followes: Column:Jupiter C18 (Phenomenex), mobile phases: (A) 0.1% formic acid in waterand (B) acetonitrile, gradient: 5%-100% (B) in 2 minutes, electrosprayionization, MRM detection (triple quadrupole). The peak areas weredetermined and triplicate values are averaged. The binding is expressedin percent of the (filtered and not-filtered time point 0 min) control 1and 2 by: 100−(100*T₆₀/T₀). The average from these values is thencalculated (see ref. 9 below).

3.0 In Vivo Studies 3.1. Maximum Tolerated Dose in Mice

a) The compound of Example 1, dispersed in Water for Injection or 0.9%physiological saline), was administered, in the preliminary study, byi.v. injection at dose levels of 35, 50, 70, 85, 100, 150, 250 or 500mg/kg to groups consisting of one male and one female mouse(Crl:CD1(ICR)). In addition, two groups comprising two male and twofemale mice received dose levels of 90 and 100 mg/kg, respectively, andthe 50 mg/kg dose level was repeated in a group comprising one male andone female.

b) Maximum tolerated dose studies (MTD) carried out with the compound ofExample 2 using CD1 mice (3 mice/group) and was performed using i.v, ipand sc. administration.

3.2 Repeated Toxicity Studies in Mice

The toxicity and toxicokinetics of the compound of Example 1 wasinvestigated following daily i.v. injection in the mouse for at least 14days. Groups of 12 male and 12 female Crl:CD1(ICR) mice received dosepreparations containing control article (50 mM sodium dihydrogenorthophosphate buffer containing 0.9% w/v sodium chloride) or 8, 24, or40 mg/kg/day POL6326 at a dose volume of 5 mL/kg. Satellite groups of 24animals per sex per group were included at each dose level. Assessmentof toxicity was based on mortality, clinical signs, body weights, foodconsumption, ophthalmic examinations, clinical and anatomic pathology,and toxicokinetic evaluations.

33 Stemcell Mobilisation a) Mice Model:

The aim of the study was to evaluate the ability of the compound ofExample 1 and Example 2 to mobilize hematopoietic progenitors from mousebone marrow to peripheral blood using in-vitro hematopoietic colonyassays. In humans, accurate information about progenitor cellmobilization is provided by the colony forming unit granulocyte-monocyte(CFU-GM) assay or by determining the abundance of CD34(+) cells by FACSanalysis (see ref. 10 below). In mice, CD34 is not a useful marker forstem cells; instead the CFU-GM is more commonly used (see ref. 11below).

In order to assess the ability of the compounds of Example 1 and Example2 to mobilize murine stem cells (CFU-GM) C3H/HeJ female mice (JacksonLaboratory) were injected s.c. with 5 mg/k, of the compounds of Example1 and of Example 2 and as reference AMD3100 (Broxineyer, et al, J ExpMed 201, 1307-1318), (currently undergoing Phase III clinical trials)for stem cell mobilisation. Peripheral blood samples from 5 animals pertest group were collected at each time point and nucleated cell countsperformed as standard assays.

b) Monkey Model:

An assessment of mobilization of peripheral-blood hematopoietic stemcells in cynomolgus monkeys (Macaca fascicularis) was performed. Thecompound of Example 1 was administred to 4 monkeys (2 male and 2 female)as a slow bolus i.v. injection over 2 minutes and CD34(+) cells weredetermined by FACS analysis. Toxicokinetic blood sampling was alsoperformed.

4.0. Results

The results of the experiments described under 2.2-2.8, above, areindicated in the following Tables 1 and 2.

TABLE 1 Cyto-toxicity IC₅₀ (μM) IC₅₀ (nM) FIGS LC₅₀/GI₅₀ Hemo-lysis Cellmigration Ex. Ca²⁺ assay IC50 (nM) Hela cells at 100 μM assay 1 5.5n.d. >50 0.6 n.d. 2 4.1 24.7 94 0.3 0.5 n.d.: not determined

TABLE 2 Ex. Stability human Plasma t_(1/2) (min) Stability rat Plasmat_(1/2) (min) 1 >240 >240 2 >300 >300

The results of the experiment described in 3.1-3.3 are given hereinbelow.

4.1: MTD Study in Mice a) MTD Study, Compound of Example 1

The acute minimum lethal intravenous dose level of Ex. 1 in the mousewas found to exceed 90 mg/kg.

b) MTD Study, Compound of Example 2

The highest dose tested for all three routes of administration was 120mg/kg bolus. At this dose all animals survived and only mild symptomswere observed. The symptoms exhibited were slight behavioral depression,slight cyanosis, an increase in respiratory depth and muscle relaxation.

4.2: 14-Day Intravenous Injection Toxicity and Toxicokinetic Study

The NOAEL level for the compound of Example 1 following i.v. dosing inthe mouse was 40 mg/kg/day.

There was no notable effect of treatment on body weight, body weightchange, or food consumption, or on ophthalmic observations during thefinal week of the dosing phase. Administration of the compound ofExample 1 was associated with mildly higher white blood cell andabsolute lymphocyte counts for females given 40 mg/kg/day. Males given40 mg/kg/day were not similarly affected, and these minor effects werenot considered adverse. Clinical chemistry results were unaffected byadministration of the compound of Example 1. Increases in organ weights(kidneys in males given 8 mg/kg/day and seminal vesicles in males given24 or 40 mg/kg/day) were considered incidental and unrelated totreatment. No test article-related gross lesions were recorded. Threeanimals (1 control group female, 1 male at 8 mg/kg/day and 1 female at24 mg/kg/day) had a focal, red, crusted area at the injection site(tail), which was the result of hypodermic needle punctures. No testarticle-related microscopic lesions were observed.

4.3: Stemcell Mobilisation a) Stem Cell Mobilization in Mice, Compoundof Example 1:

Administration of 5 mg/kg of the compound of Example 1 increased theCFU-GM blood cell numbers with a maximal effect at 120 minutes andreturn to baseline levels 6 h post administration. (FIG. 1). In the sameassay, AMD3100 (currently undergoing Phase III clinical trials for stemcell mobilisation) was used as a comparator. In a follow up study, thedose response of Ex. 1 on the release of CFU-GM was determined (FIG. 1).There is a clear dose response effect of Ex. 1 on the release of CFU-GMin mice with a peak level increase at 5 mg/kg.

b) Stem Cell Mobilization in Mice, Compound of Example 2:

Administration of 5 mg/kg of the compound of Example 2 increased theCFU-GM blood cell numbers up to six hours post administration with amaximal effect at 240 minutes whereas administration of AMD3100 isassociated with an increase in the frequency and number of progenitorsat 30 and 60 minutes compared to control mice (FIG. 2).

c) Stem Cell Mobilization in Monkey, Compound of Example 1:

Administration of the compound of Example 1 induced mobilization ofCD34(+) hematopoietic cells in cynomolgus monkeys. As observed in mice,the onset of mobilization was rapid with a peak level at two hours. Themobilisation was also transient and the numbers of stem cells inperipheral blood returned to baseline level with decreasing plasmalevels of the compound of Example 1 (FIG. 3).

REFERENCES

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1-9. (canceled)
 10. A method for treating cancer that expresses a CXCR4receptor, the method comprising: administering a therapeuticallyeffective amount of aCyclo(-Tyr-His-X-Cys-Ser-Ala-^(D)Pro-Dab-Ara-Tyr-Cys-Tyr-Gln-Lys-^(D)Pro-Pro)compound having a disulfide bond between Cys4 and Cys11, or apharmaceutically acceptable salt thereof, wherein X is Ala or Tyr to asubject in need thereof.
 11. The method of claim 10, wherein theadministering is oral, topical, transdermal, injection, buccal,transmucosal, pulmonary or inhalation administration.
 12. The method ofclaim 10, wherein the compound is administered in a composition furthercomprising a pharmaceutically inert carrier and wherein the compositionis in the form of a tablet, a dragee, a capsule, a solution, a liquid, agel, a plaster, a cream, an ointment, a syrup, a slurry, a suspension, aspray, a nebulizer, or a suppository.
 13. The method of claim 10,wherein the cancer is breast cancer.
 14. The method of claim 10, whereinX is Ala.